Hydrolyzed proteins, which are widely used in the food industry, may be prepared by hydrolysis of protein material with acid, alkali or enzymes. Various methods have been used koji molds for the preparation food products, which are hydrolyzed by action of a large variety of secreted amylases, proteinases and peptidases. Koji molds are those traditionally used for making a koji culture (U.S. Pat. No. 4,308,284) including cells of the genus Aspergillus, Rhizopus and/or Mucor, especially Aspergillus soyae, Aspergillus oryzae, Aspergillus phoenicis, Aspergillus niger, Aspergillus awamori, Rhizopus oryzae, Rhizopus oligosporus, Rhizopus japonicus, Rhizopus formosaensis, Mucor circinelloides, Mucor japanicus, Penicillium glaucum and Penicillium fuscum, for example.
According to the rules of the International Code of Botanical Nomenclature (ICBN), Aspergillus is an anamorphic genus. This means that true Aspergillus only reproduce asexually through conidiophores. However, the typical Aspergillus conidiophore morphology can also be found in fungi that can reproduce sexually via ascospores. Some Aspergillus taxonomists caused confusion, because they did not adhere to ICBN terminology. Instead, they attempted to make various revisions of taxonomical schemes to include Aspergillus nidulans in this genus, despite the fact that its taxonomically correct name is Emericelia nidulans (Samson, In: Aspergillus. Biology and Industrial Applications, pp 355-390, Ed. by Bennett and Klich, Boston)
EP417481 (Societe des Produits Nestle) thus describes a process for the production of a fermented soya sauce, in which a koji is prepared by mixing a koji culture with a mixture of cooked soya and roasted wheat, the koji is then hydrolyzed in aqueous suspension for 3 to 8 hours at 45.degree. C. to 60.degree. C. with the enzymes produced during fermentation of the koji culture, a moromi is further prepared by adding sodium chloride to the hydrolyzed koji suspension, the moromi is left to ferment and is then pressed and the liquor obtained is pasteurized and clarified.
EP429760 (Societe des Produits Nestle) describes a process for the production of a flavoring agent in which an aqueous suspension of a protein-rich material is prepared, the proteins are solubilized by hydrolysis of the suspension with a protease at pH6.0 to 11.0, the suspension is heat-treated at pH 4.6 to 6.5, and the suspension is ripened with enzymes of a koji culture.
Likewise, EP96201923.8 (Societe des Produits Nestle) describes a process for the production of a meat flavor, in which a mixture containing a vegetal proteinaceous source and a vegetal carbohydrates containing source is prepared, said mixture having initially at least 45% dry matter, the mixture is inoculated with a koji culture and by one or more another species of microorganisms involved in the traditional fermentation of meat, and the mixture is incubated until meat flavors are formed.
However, on the one hand, acid or alkaline hydrolysis can destroy the essential amino acids produced during hydrolysis thus reducing the nutritional value, whereas enzymatic hydrolysis rarely goes to completion so that the hydrolyzed protein contains substantial amounts of peptides. The optimization and further development of koji processes have been seriously hampered by the lack of knowledge on the nature of the hydrolytic enzymes, their regulation and how process parameters affect their expression and activity (e.g. temperature, pH, water activity, and salt concentration).
In the fungal Emericella nidulans (Katz et al., Gene, 150, 287-292, 1994), fermentation activity is subject to at least three general control circuits including carbon catabolite repression, nitrogen and sulfur metabolite repression. These three regulatory circuits ensure that the available nitrogen-, carbon-, and sulfur sources in a substrate are utilized sequentially according to their nitrogen, energy and sulfur yield. Nitrogen metabolite repression is exerted by the areA gene product in Emericella nidulans (Arst et al., Mol. Gen. Genet., 26, 111-141, 1973), whereas in the other fungals Neurospora crassa (Davies et al., Proc. Natl. Acad. Sci. USA, 84, 3753-3757, 1987), Penicillium chrysogenum (Haas et al., Curr. Genet., 27, 150-158, 1995) and Saccharomyces cerevisiae (Minehart et al., Mol. Cell. Biol., 11, 6216-6228, 1991) similar genes exert a similar function.
The areA gene encodes a positively acting DNA-binding protein (AREA), belonging to the GATA family of transcription factors, that is required for the utilization of all nitrogen sources except ammonia or L-glutamine. Under nitrogen de-repressed conditions, signaled by high intracellular levels of glutamine, areA expression is down regulated by three mechanisms: 1) the AREA protein is inactivated, 2) areA transcription is halted and 3) by action of the 3' untranslated trailer sequence (3'-UTS) areA mRNA degradation is enhanced (Platt et al., EMBO J., 15, 2791-2801, 1996). In the absence of a functional AREA protein, only ammonia or L-glutamine can be utilized as nitrogen source. Consequently, loss-of-function areA mutants can utilize only ammonia or L-glutamine as nitrogen sources (Arst et al., 1973).
Observations in koji fermentation suggest that nitrogen metabolite repression is a major parameter in koji fermentation. For instance, high levels of L-glutamine are shown to negatively affect proteolytic activity in koji fermentation.
Furthermore, it has been observed that high levels of proteolytic activity and glutaminase activity are two mutually exclusive conditions in koji fermentation (Ushijima et al., Agric. Biol. Chem., 51, 1051-1057, 1997). For instance, addition of 25 mM L-glutamine into a minimal growth medium containing 0.1% wheat gluten reduces endoproteolytic enzyme activity about 40-50 fold. This phenomenon may be explained by postulating that L-glutamine is necessary for the induction of glutaminase. However, since L-glutamine is also the effector of nitrogen metabolite repression, the expression of proteolytic enzymes is suppressed when glutaminase is induced.
With regard to the fact that glutaminase suitably converts L-glutamine into L-glutamic acid which is an important natural taste enhancer (see WO95/31114), there is hence a need to overcome L-glutamine mediated suppression of proteolytic enzymes, allowing simultaneous expression of glutaminase and proteolytic enzymes in koji molds.
In addition, depending on the nature of the protein and the enzymes used for proteolysis, the peptides formed can however have extremely bitter tastes and are thus organoleptically undesirable. There is hence also a need for methods of hydrolyzing proteins leading to high degree of protein hydrolysis and to hydrolysates with excellent organoleptic properties.
Finally, biochemical analysis of residual peptides in cereals hydrolyzed by koji molds, e.g. wheat gluten, shows that a considerable amount of L-glutamine remains sequestered in proline containing peptides (Adler-Nissen, In: Enzymatic hydrolysis of food proteins. Elsevier Applied Sciences Publishers LTD, p120, 1986). There is hence also a need for methods of hydrolyzing proteins leading to liberation of high amount of L-glutamine.